Cyclic and substituted immobilized molecular synthesis

ABSTRACT

A synthetic strategy for the creation of large scale chemical diversity. Solid-phase chemistry, photolabile protecting groups, and photolithography are used to achieve light-directed spatially-addressable parallel chemical synthesis. In one particular embodiment, an array of rotated cyclic polymers is formed. In another embodiment, an array of polymers is formed based on a target polymer. The array includes systematically substituted versions of the target molecule. In another embodiment, rotated and systematically substituted cyclic polymers are formed on a substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 796,727, filed Nov. 22, 1991. This application is also acontinuation-in-part of U.S. application Ser. No. 805,727, filed Dec. 6,1991, which is a continuation-in-part of Ser. No. 624,120, filed Dec. 6,1990, which is a continuation-in-part of application Ser. No. 492,462(now U.S. Pat. No. 5,143,854), filed Mar. 7, 1990, which is acontinuation-in-part of U.S. application Ser. No. 362,901, filed Jun. 7,1989 (abandoned). All of the above identified applications areincorporated herein by reference for all purposes.

[0002] This application is also related to the following United Statesapplications: U.S. application Ser. Nos. 626,730 and 624,114, both filedDec. 6, 1990; and U.S. application Ser. Nos. 796,243 and 796,947, bothfiled on Nov. 22, 1991. Each of these applications is incorporatedherein by reference for all purposes.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to the field of molecularsynthesis. More specifically, the invention provides systems and methodsfor directed synthesis of diverse molecular sequences on substrates.

[0004] Methods for preparing different polymers are well known. Forexample, the “Merrifield” method, described in Atherton et al., “SolidPhase Peptide Synthesis,” IRL Press, 1989, which is incorporated hereinby reference for all purposes, has been used to synthesize peptides on asolid support. In the Merrifield method, an amino acid is covalentlybonded to a support made of an insoluble polymer or other material.Another amino acid with an alpha protecting group is reacted with thecovalently bonded amino acid to-form a dipeptide. After washing, theprotecting group is removed and a third amino acid with an alphaprotecting group is added to the dipeptide. This process is continueduntil a peptide of a desired length and sequence is obtained.

[0005] Other techniques have also been described. These methods includethe synthesis of peptides on 96 plastic pins which fit the format ofstandard microtiter plates. Advanced techniques for synthesizing largenumbers of molecules in an efficient manner have also been disclosed.Most notably, U.S. Pat. No. 5,143,854 (Pirrung et al.) and PCTApplication No. 92/10092 disclose improved methods of molecularsynthesis using light directed techniques. According to these methods,light is directed to selected regions of a substrate to removeprotecting groups from the selected regions of the substrate.Thereafter, selected molecules are coupled to the substrate, followed byadditional irradiation and coupling. steps.

SUMMARY OF THE INVENTION

[0006] Methods, devices, and compositions for synthesis and use ofdiverse molecular sequences on a substrate are disclosed, as well asapplications thereof.

[0007] A preferred embodiment of the invention provides for thesynthesis of an array of polymers in which individual monomers in a leadpolymer are systematically substituted with monomers from one or morebasis sets of monomers. The method requires a limited number of masksand a limited number of processing steps. According to one specificaspect of the invention, a series of masking steps are conducted tofirst place the first monomer in the lead sequence on a substrate at aplurality of synthesis sites. The second monomer in the lead sequence isthen added to the first monomer at a portion of the synthesis sites,while different monomers from a basis set are placed at discrete othersynthesis sites. The process is repeated to produce all or a significantnumber of the mono substituted polymers based on the lead polymer usinga given basis set of monomers. According to a preferred aspect of theinvention, the technique uses light directed techniques, such as thosedescribed in Pirrung et al., U.S. Pat. No. 5,143,854.

[0008] Another aspect of the invention provides for efficient synthesisand screening of cyclic molecules. According to a preferred aspect ofthe invention, cyclic polymers are synthesized in an array in which thepolymers are coupled to the substrate at different positions on thecyclic polymer ring. Therefore, a particular polymer may be presented invarious “rotated” forms on the substrate for later screening. Again, thecyclic polymers are formed according to most preferred embodiments withthe techniques of Pirrung et al.

[0009] The resulting substrates will have a variety of uses including,for example, screening polymers for biological activity. To screen forbiological activity, the substrate is exposed to one or more receptorssuch as an antibody, oligonucleotide, whole cells, receptors onvesicles, lipids, or any one of a variety of other receptors. Thereceptors are preferably labeled with, for example, a fluorescentmarker, a radioactive marker, or a labeled antibody reactive with thereceptor. The location of the marker on the substrate is detected with,for example, photon detection or auto-radiographic techniques. Throughknowledge of the sequence of the material at the location where bindingis detected, it is possible to quickly determine which polymer(s) arecomplementary with the receptor. The technique can be used to screenlarge numbers of peptides or other polymers quickly and economically.

[0010] A further understanding of the nature and advantages of theinventions herein may be realized by reference to the remaining portionsof the specification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIGS. 1A to 1C illustrate a systematic substitution maskingstrategy;

[0012]FIG. 2 illustrates additional aspects of a systemmaticsubstitution masking strategy;

[0013]FIGS. 3, 4, and 5 illustrate rotated cyclic polymer groups;

[0014]FIGS. 6A to 6E illustrate formation of rotated cyclic polymers;

[0015]FIGS. 7A to 7C illustrate formation of rotated and substitutedcyclic polymers;

[0016]FIG. 8 illustrates the array of cyclic polymers resulting from thesynthesis;

[0017]FIG. 9 illustrates masks used in the synthesis of cyclic polymerarrays;

[0018]FIGS. 10A and 10B illustrate coupling of a tether in twoorientations;

[0019]FIGS. 11A and 11B illustrate masks used in another embodiment;

[0020]FIGS. 12A and 12B show a tripeptide used in a fluorescenceenergy-transfer substrate assay and that substrate after cleavage;

[0021]FIGS. 13A to 13H illustrate donor/quencher pairs;

[0022]FIG. 14 illustrates sequence versus normalized fluorescenceintensity for the ten possible single deletion peptides binding to theD32.39 antibody. A blank space represents a deleted amino acid relativeto the full length kernel sequence (FLRRQFKVVT) shown on the bottom.Error bars represent the standard deviation of the averaged signals fromfour replicates. All peptides are acetylated on the amino terminus andare linked to the surface via an amide bond to the carboxyl terminus;and

[0023]FIG. 15 illustrates sequence versus normalized fluorescenceintensity for the terminally truncated peptides. The full length kernalsequence (FLRQFKVVT) is shown in the center of the graph. Error barsrepresent the standard deviation of the averaged signals from a minumumof four replicates. All peptides are acetylated on the amino terminusand are linked to the surface via an amide bond to the carboxylterminus.

DESCRIPTION OF THE PREFERRED EMBODIMENTS CONTENTS

[0024] I. Definitions

[0025] II. Synthesis

[0026] A. Systematic Substitution

[0027] B. Cyclic Polymer Mapping

[0028] III. Data Collection

[0029] A. CCD Data Collection System

[0030] B. Trapping Low Affinity Interactions

[0031] C. Fluorescence Energy-Transfer Substrate Assays

[0032] IV. Examples

[0033] A. Example

[0034] B. Example

[0035] V. Conclusion

[0036] I. Definitions

[0037] Certain terms used herein are intended to have the followinggeneral definitions:

[0038] 1. Complementary: This term refers to the topologicalcompatibility or matching together of interacting surfaces of a ligandmolecule and its receptor. Thus, the receptor and its ligand can bedescribed as complementary, and furthermore, the contact surfacecharacteristics are complementary to each other.

[0039] 2. Epitope: An epitope is that portion of an antigen moleculewhich is delineated by the area of interaction with the subclass ofreceptors known as antibodies.

[0040] 3. Ligand: A ligand is a molecule that is recognized by aparticular receptor. Examples of ligands that can be investigated bythis invention include, but are not restricted to, agonists andantagonists for cell membrane receptors, toxins and venoms, viralepitopes, hormones, hormone receptors, peptides, enzymes, enzymesubstrates, cofactors, drugs (e.g., opiates, steroids, etc.), lectins,sugars, oligonucleotides (such as in hybridization studies), nucleicacids, oligosaccharides, proteins, benzodiazapines, prostoglandins,beta-turn mimetics, and monoclonal antibodies.

[0041] 4. Monomer: A monomer is a member of the set of smaller moleculeswhich can be joined together to form a larger molecule. The set ofmonomers includes but is not restricted to, for example, the set ofcommon L-amino acids, the set of D-amino acids, the set of natural orsynthetic amino acids, the set of nucleotides and the set of pentosesand hexoses. As used herein, monomer refers to any member of a basis setfor synthesis of a larger molecule. A selected set of monomers forms abasis set of monomers. For example, dimers of the 20 naturally occurringL-amino acids form a basis set of 400 monomers for synthesis ofpolypeptides. Different basis sets of monomers may be used in any of thesuccessive steps in the synthesis of a polymer. Furthermore, each of thesets may include protected members which are modified after synthesis.

[0042] 5. Peptide: A peptide is a polymer in which the monomers arenatural or unnatural amino acids and which are joined together throughamide bonds, alternatively referred to as a polypeptide. In the contextof this specification, it should be appreciated that the amino acids maybe, for example, the L-optical isomer or the D-optical isomer. Specificimplementations of the present invention will result in the formation ofpeptides with two or more amino acid monomers, often 4 or more aminoacids, often 5 or more amino acids, often 10 or more amino acids, often15 or more amino acids, and often 20 or more amino acid. Standardabbreviations for amino acids are used (e.g., P for proline). Theseabbreviations are included in Stryer, Biochemistry, Third Ed., 1988,which is incorporated herein by reference for all purposes.

[0043] 6. Radiation: Radiation is energy which may be selectivelyapplied, including energy having a wavelength of between 10⁻¹⁴ and 10⁴meters including, for example, electron beam radiation, gamma radiation,x-ray radiation, light such as ultra-violet light, visible light, andinfrared light, microwave radiation, and radio waves. “Irradiation”refers to the application of radiation to a surface.

[0044] 7. Receptor: A receptor is a molecule that has an affinity for agiven ligand. Receptors may be naturally-occurring or syntheticmolecules. Also, they can be employed in their unaltered state, inderivative forms, or as aggregates with other species. Receptors may beattached, covalently or noncovalently, to a binding member, eitherdirectly or via a specific binding substance. Examples of receptorswhich can be employed by this invention include, but are not restrictedto, antibodies, cell membrane receptors, monoclonal antibodies andantisera reactive with specific antigenic determinants (such as onviruses, cells, or other materials), drugs, oligonucleotides,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the termreceptors is used herein, no difference in meaning is intended. A“Ligand Receptor Pair” is formed when two molecules have combinedthrough molecular recognition to form a complex.

[0045] Other examples of receptors which can be investigated by thisinvention include but are not restricted to microorganism receptors,enzymes, catalytic polypeptides, hormone receptors, and opiatereceptors.

[0046] 8. Substrate: A substrate is a material having a rigid orsemi-rigid surface, generally insoluble in a solvent of interest such aswater, porous and/or non-porous. In many embodiments, at least onesurface of the substrate will be substantially flat, although in someembodiments it may be desirable to physically separate synthesis regionsfor different polymers with, for example, wells, raised regions, etchedtrenches, or the like. According to other embodiments, small beads maybe provided on the surface which may be released upon completion of thesynthesis.

[0047] 9. Protecting group: A protecting group is a material which ischemically bound to a monomer unit or polymer and which may be removedupon selective exposure to an activator such as electromagneticradiation or light, especially ultraviolet and visible light. Examplesof protecting groups with utility herein include those comprisingortho-nitro benzyl derivatives, nitropiperonyl, pyrenylmethoxy-carbonyl,nitroveratryl, nitrobenzyl, dimethyl dimethoxybenzyl,5-bromo-7-nitroindolinyl, o-hydroxy-α-methyl cinnamoyl, and2-oxymethylene anthraquinone.

[0048] 10. Predefined Region: A predefined region is a localized area ona surface which is, was, or is intended to be activated for formation ofa molecule using the techniques described herein. The predefined regionmay have any convenient shape, e.g., circular, rectangular, elliptical,wedge-shaped, etc. For the sake of brevity herein, “predefined regions”are sometimes referred to simply as “regions.” A predefined region maybe illuminated in a specified step, along with other regions of asubstrate.

[0049] 11. Substantially Pure: A molecule is considered to be“substantially pure” within a predefined region of a substrate when itexhibits characteristics that distinguish it from other predefinedregions. Typically, purity will be measured in terms of biologicalactivity or function as a result of uniform sequence. Suchcharacteristics will typically be measured by way of binding with aselected ligand or receptor. Preferably the region is sufficiently puresuch that the predominant species in the predefined region is thedesired sequence. According to preferred aspects of the invention, themolecules formed are 5% pure, more preferably more than 10% pure,preferably more than 20% pure, more preferably more than 80% pure, morepreferably more than 90% pure, more preferably more than 95% pure, wherepurity for this purpose refers to the ratio of the number of ligandmolecules formed in a predefined region having a desired sequence to thetotal number of molecules formed in the predefined region.

[0050] 12. Activator: A activator is a material or energy source adaptedto render a group active and which is directed from a source to at leasta predefined location on a substrate, such as radiation. A primaryillustration of an activator is light, such as visible, ultraviolet, orinfrared light. Other examples of activators include ion beams, electricfields, magnetic fields, electron beams, x-ray, and the like.

[0051] 13. Combinatorial Synthesis Strategy: A combinatorial synthesisstrategy is an ordered strategy for parallel synthesis of diversepolymer sequences by sequential addition of reagents which may berepresented by a reactant matrix and a switch matrix, the product ofwhich is a product matrix. A reactant matrix is a l column by m rowmatrix of the building blocks to be added. The switch matrix is all or asubset of the binary numbers, preferably ordered, between 1 and marranged in columns. A “binary strategy” is one in which at least twosuccessive steps illuminate a portion, often half, of a region ofinterest on the substrate. In a binary synthesis strategy, all possiblecompounds which can be formed from an ordered set of reactants areformed. In most preferred embodiments, binary synthesis refers to asynthesis strategy which also factors a previous addition step. Forexample, a strategy in which a switch matrix for a masking strategyhalves regions that were previously illuminated, illuminating about halfof the previously illuminated region and protecting the remaining half(while also protecting about half of previously protected regions andilluminating about half of previously protected regions). It will berecognized that binary rounds may be interspersed with non-binary roundsand that only a portion of a substrate may be subjected to a binaryscheme. A combinatorial “masking” strategy is a synthesis which useslight or other spatially selective deprotecting or activating agents toremove protecting groups from materials for addition of other materialssuch as amino acids.

[0052] 14. Linker: A linker is a molecule or group of molecules attachedto a substrate and spacing a synthesized polymer from the substrate forexposure/binding to a receptor.

[0053] 15. Systematically Substituted: A position in a target moleculehas been systematically substituted when the molecule is formed at aplurality of synthesis sites, with the molecule having a differentmember of a basis set of monomers at the selected position of themolecule within each of the synthesis sites on the substrate.

[0054] 16. Abbreviations: The following frequently used abbreviationsare intended to have the following meanings:

[0055] BOC: t-butyloxycarbonyl.

[0056] BOP: benzotriazol-1-yloxytris-(dimethylamino) phosphoniumhexafluorophosphate.

[0057] DCC: dicyclohexylcarbodiimide.

[0058] DCM: dichloromethane; methylene chloride.

[0059] DDZ: dimethoxydimethylbenzyloxy.

[0060] DIEA: N,N-diisopropylethylamine.

[0061] DMAP: 4-dimethylaminopyridine.

[0062] DMF: dimethyl formamide.

[0063] DMT: dimethoxytrityl.

[0064] FMOC: fluorenylmethyloxycarbonyl.

[0065] HBTU: 2-(1H-benzotriazol-1-y1)-1,1,3,3-tetramethyluroniumhexafluorophosphate.

[0066] HOBT: 1-hydroxybenzotriazole.

[0067] NBOC: 2-nitrobenzyloxycarbonyl.

[0068] NMP: N-methylpyrrolidone.

[0069] NPOC: 6-nitropiperonyloxycarbonyl.

[0070] NV: 6-nitroveratryl.

[0071] NVOC: 6-nitroveratryloxycarbonyl.

[0072] PG: protecting group.

[0073] TFA: trifluoracetic acid.

[0074] THF: tetrahydrofuran.

[0075] II. Synthesis

[0076] The present invention provides synthetic strategies and devicesfor the creation of large scale chemical diversity. Solid-phasechemistry, photolabile protecting groups, and photolithography arebrought together to achieve light-directed spatially-addressableparallel chemical synthesis in preferred embodiments.

[0077] The invention is described herein for purposes of illustrationprimarily with regard to the preparation of peptides and nucleotides butcould readily be applied in the preparation of other molecules. Suchmolecules include, for example, both linear and cyclic polymers ofnucleic acids, polysaccharides, phospholipids, and peptides havingeither α-, β-, or ω-amino acids, heteropolymers in which a known drug iscovalently bound to any of the above, poly-urethanes, polyesters,polycarbonates, polyureas, n-alkylureas, polyamides, polyethyleneimines,polyarylene sulfides, polysiloxanes, polyimides, carbamates, sulfones,sulfoxides, polyacetates, or other polymers which will be apparent uponreview of this disclosure. It will be recognized further that peptideillustrations herein are primarily with reference to C- to N-terminalsynthesis, but the invention could readily be applied to N- toC-terminal synthesis without departing from the scope of the invention.Methods for forming cyclic and reversed polarity peptides and otherpolymers are described in copending application Ser. No. 796,727, filedNov. 22, 1991, and previously incorporated herein by reference. Othermolecules that are not conventionally viewed as polymers but which areformed from a basis set of monomers or building blocks may also beformed according to the invention herein.

[0078] The prepared substrate may, for example, be used in screening avariety of polymers as ligands for binding with a receptor, although itwill be apparent that the invention could be used for the synthesis of areceptor for binding with a ligand. The substrate disclosed herein willhave a wide variety of uses. Merely by way of example, the inventionherein can be used in determining peptide and nucleic acid sequencesthat bind to proteins, finding sequence-specific binding drugs,identifying epitopes recognized by antibodies, and evaluating a varietyof drugs for clinical and diagnostic applications, as well ascombinations of the above.

[0079] The invention preferably provides for the use of a substrate “S”with a surface. Linker molecules “L” are optionally provided on asurface of the substrate. The purpose of the linker molecules, in someembodiments, is to facilitate receptor recognition of the synthesizedpolymers.

[0080] Optionally, the linker molecules are chemically protected forstorage or synthesis purposes. A chemical protecting group such as t-BOC(t-butyloxycarbonyl) is used in some embodiments. Such chemicalprotecting groups would be chemically removed upon exposure to, forexample, acidic solution and could serve, inter alia to protect thesurface during storage and be removed prior to polymer preparation.

[0081] When a polymer sequence to be synthesized is, for example, apolypeptide, amino groups at the ends of linkers attached to a glasssubstrate are derivatized with, for example, nitroveratryloxycarbonyl(NVOC), a photoremovable protecting group. The linker molecules may be,for example, aryl acetylene, ethylene glycol oligomers containing from2-10 monomers, diamines, diacids, amino acids, or combinations thereof.

[0082] According to one aspect of the invention, on the substrate or adistal end of the linker molecules, a functional group with a protectinggroup P₀ is provided. The protecting group P₀ may be removed uponexposure to an activator such as a chemical reagent, radiation, electricfields, electric currents, or other activators to expose the functionalgroup. In a preferred embodiment, the radiation is ultraviolet (UV),infrared (IR), or visible light, or a basic or acidic reagent. In stillfurther alternative embodiments, ion beams, electron beams, or the likemay be used for deprotection.

[0083] Photodeprotection is effected by illumination of the substratethrough, for example, a mask wherein the pattern produces illuminatedregions with dimensions of, for example, less than 1 cm² , 10⁻¹ cm²,10⁻² cm², 10⁻³ cm² , 10⁻⁴ cm², 10⁻⁵ cm², 10⁻⁶ cm² , 10⁻⁷ cm² , 10⁻⁸ cm²,or 10⁻¹⁰ cm². In a preferred embodiment, the regions are between about10×10 μm and 500×500 μm. According to some embodiments, the masks arearranged to produce a checkerboard array of polymers, although any oneof a variety of geometric configurations may be utilized.

[0084] Concurrently with or after exposure of a known region of thesubstrate to light or another activator, the surface is contacted with afirst monomer unit M₁ which reacts with the functional group that hasbeen exposed by the deprotection step. The first monomer includes aprotecting group P₁, P₁, may or may not be the same as P₀.

[0085] Accordingly, after a first cycle, first regions of the surfacecomprise the sequence:

S—L—M₁—P₁

[0086] while remaining regions of the surface comprise the sequence:

S—L—P₀ .

[0087] Thereafter, one or more second regions of the surface (which mayinclude all or part of the first region, as well as other regions) areexposed to light and contacted with a second monomer M₂ (which may ormay not be the same as M₁) having a protecting group P₂. P₂ may or maynot be the same as P₀ and P₁. After this second cycle, different regionsof the substrate may comprise one or more of the following sequences:

S—L—M₁—M₂—P₂

S—L—M₂—P₂

S—L—M₁—P₁ and/or

S—L—P₀.

[0088] The above process is repeated until the substrate includesdesired polymers of desired lengths. By controlling the locations of thesubstrate exposed to light and the reagents exposed to the substratefollowing exposure, one knows the location of each sequence.

[0089] According to some embodiments of the invention, multipleprotecting groups are utilized. For example, when light-labileprotecting groups are utilized to protect the growing polymer chain, itwill be desirable in some embodiments to provide different protectinggroups on at least selected side groups of the various monomers. Forexample, acid or base labile protecting groups may be particularlydesirable when light labile protecting groups are used on the growingpolymer chain. As a specific example, in the case of amino acids, thesulfhydryl groups of cysteine side chains can form disulfide bonds withone another. Accordingly, it will sometimes be desirable to protect suchside groups with an acid or base labile protecting group, or aprotecting group that is removed with a wavelength of light differentfrom that which removes the protecting group on the growing polymerchain. Then, one can selectively couple these side chains by removingthe appropriate protecting groups.

[0090] Thereafter, the protecting groups are removed from some or all ofthe substrate and the sequences are, optionally, capped with a cappingunit C. The process results in a substrate having a surface with aplurality of polymers of the following general formula:

S—n [L]—(M_(i))—(M_(j))—(M_(k)) . . . (M_(x))—[C]

[0091] where square brackets indicate optional groups, and M_(i). . .M_(x) indicates any sequence of monomers. The number of monomers couldcover a wide variety of values, but in a preferred embodiment they willrange from 2 to 100.

[0092] In some embodiments, a plurality of locations on the substratepolymers contain a common monomer subsequence. For example, it may bedesired to synthesize a sequence S—M₁—M₂—M₃ at first locations and asequence S—M₄—M₂—M₃ at second locations. The process would commence withirradiation of the first locations followed by contacting with M₁—P,resulting in the sequence S—M₁—P at the first location. The secondlocations would then be irradiated and contacted with M₄—P, resulting inthe sequence S—M₄—P at the second locations. Thereafter both the firstand second locations would be irradiated and contacted with monomers M₂and M₃ (or with the dimer M₂—M₃, resulting in the sequence S—M₁—M₂—M₃ atthe first locations and S—M₄—M₂—M₃ at the second locations. Of course,common subsequences of any length could be utilized including those in arange of 2 or more monomers, such as 2 to 10 monomers, 2 to 20 monomers,or 2 to 100 monomers.

[0093] The polymers prepared on a substrate according to the abovemethods will have a variety of uses including, for example, screeningfor biological activity, i.e., such as ability to bind to a receptor. Insuch screening activities, the substrate containing the sequences isexposed to an unlabeled or labeled receptor such as an antibody, areceptor on a cell, a phospholipid vesicle, or any one of a variety ofother receptors. In one preferred embodiment, the polymers are exposedto a first, unlabeled or labeled receptor of interest and thereafterexposed to a labeled receptor-specific recognition element, which is,for example, an antibody. This process can provide signal amplificationin the detection stage.

[0094] The receptor molecules may or may not bind with one or morepolymers on the substrate. The presence (or lack thereof) of the labeledreceptor and, therefore, the presence of a sequence which binds with thereceptor is detected in a preferred embodiment through the use ofautoradiography, detection of fluorescence with a charge-coupled device,fluorescence microscopy, or the like. The sequence of the polymer at thelocations where the receptor binding is detected may be used todetermine all or part of a sequence which is complementary to thereceptor.

[0095] Use of the invention herein is illustrated primarily withreference to screening for binding to a complementary receptor. Theinvention will, however, find many other uses. For example, theinvention may be used in information storage (e.g., on optical disks),production of molecular electronic devices, production of stationaryphases in separation sciences, production of dyes and brighteningagents, photography, and in immobilization of cells, proteins, lectins,nucleic acids, polysaccharides, and the like in patterns on a surfacevia molecular recognition of specific polymer sequences. By synthesizingthe same compound in adjacent, progressively differing concentrations,one can establish a gradient to control chemotaxis or to developdiagnostic “dipsticks,” which, for example, titrate an antibody againstan increasing amount of antigen. By synthesizing several catalystmolecules in close proximity, one can achieve more efficient multistepconversions by “coordinate immobilization.” Coordinate immobilizationalso may be used for electron transfer systems, as well as to provideboth structural integrity and other desirable properties to materials,such as lubrication, wetting, etc.

[0096] According to alternative embodiments, molecular biodistributionor pharmacokinetic properties may be examined. For example, to assessresistance to intestinal or serum proteases, polymers may be capped witha fluorescent tag and exposed to biological fluids of interest.

[0097] A high degree of miniaturization is possible, because the densityof compounds on the surface is determined largely with regard to spatialaddressability of the activator, in one case the diffraction of light.Each compound is physically accessible and its position is preciselyknown. Hence, the array is spatially-addressable, and its interactionswith other molecules can be assessed.

[0098] According to one aspect of the invention, reactions take place inan appropriate reaction chamber that includes isolated fluid flow pathsfor heating or cooling liquids that are used to maintain the reactionchamber temperature at a desired level. In still further embodiments thereaction chamber is placed on a rotating “centrifuge” to reduce thevolume of reactants needed for the various coupling/deprotection stepsdisclosed herein. In a centrifuge flow cell, the substrate is placed inthe centrifuge such that, for example, when a monomer solution passesover the surface of the substrate a relatively thin film of the materialis formed on the substrate due to the higher gravitational forces actingon the substrate. Accordingly, the volume of various reagents needed inthe synthesis will be substantially reduced.

[0099] A. Systematic Substitution

[0100] According to one preferred embodiment of the invention, a “lead”sequence is identified using either the light directed techniquesdescribed herein, or more conventional methods such as those describedin Geysen, J. Imm. Methods (1987) 102:259-274, incorporated herein byreference for all purposes, or through other knowledge of the structureof the receptor in question, such as through computer modelinginformation. As used herein, a “lead” or “kernel” sequence is a moleculehaving a monomer sequence which has been shown to exhibit at leastlimited binding affinity with a receptor or class of receptors.

[0101] Thereafter, a series of molecules related to the lead sequenceare generated by systematic substitution, deletion, addition, or acombination of these processes at one or more positions of the molecule.A sequence with a binding affinity higher than the lead sequence can be(or may be) identified through evaluation of the molecules produced bythese processes.

[0102] One aspect of the invention herein provides for improved methodsfor forming molecules with systematically substituted monomers or groupsof monomers using a limited number of synthesis steps. Like the otherembodiments of the invention described herein, this aspect of theinvention has applicability not only to the evaluation of peptides, butalso other molecules, such as oligonucleotides and polysaccharides.Light-directed techniques are utilized in preferred embodiments becauseof the significant savings in time, labor, and the like.

[0103] According to one aspect of the invention, a lead polymer sequenceis identified using conventional techniques or the more sophisticatedlight directed techniques described herein. The lead sequence isgenerally represented herein by:

A B C D E F G

[0104] where the various letters refer to amino acids or other monomersin their respective positions in the lead sequence. Although a polymerwith seven monomers is used herein for the purpose of illustration, alarger or smaller number of monomers will typically be found in the leadpolymer in most embodiments of the invention.

[0105] Using a selected basis set of monomers, such as twenty aminoacids or four nucleotides, one generates the following series ofsystematically substituted polymers. The sequence of the moleculesgenerated is determined with reference to the columns of the map. Inother words, the “map” below can be viewed as a cross-section of thesubstrate: G G G G G G X F F F F F X F E E E E X E E D D D X D D D C C XC C C C B X B B B B B X A A A A A A

[0106] where X represents the monomers in a basis set of monomers suchas twenty amino acids. For example, the twenty polymers XBCDEFG aregenerated within 20 individual synthesis sites on the substrate. In thecase of 7-monomer lead peptides, the total number of peptides generatedwith all twenty monomers in the basis set is 140, i.e., 7*20, with 134unique sequences being made and 7 occurences of the lead sequence.

[0107] One of the least efficient ways to form this array of polymerswould be via conventional synthesis techniques, which would requireabout 938 coupling steps (134 peptides * 7 residues each). At the otherextreme, each of these sequences could be made in 7 steps, but thesequences would be physically mixed, requiring separation afterscreening.

[0108] By contrast, this aspect of the invention provides for efficientsynthesis of substituted polymers. FIG. 1 illustrates the maskingstrategy for the 7-monomer lead polymer. The particular masking strategyillustrated in FIG. 1 utilizes rectangular masks, but it will beapparent that other shapes of masks may also be used without departingfrom the scope of the invention herein. Masking techniques whereinregions of a substrate are selectively activated by light are describedherein by way of a preferred embodiment. The inventions herein are notso limited, however, and other activation techniques may be utilized.For example, mechanical techniques of activation/coupling such asdescribed in copending application Ser. No. 07/796,243 are used in someembodiments.

[0109] As shown in FIG. 1A, the process begins by exposing substantiallyall of a predefined region of the substrate to light with a mask 291,exposing approximately {fraction (6/7)} of the region of interest 289.This step is followed by exposure of the substrate to monomer A.Thereafter, a mask 292 is used to expose approximately {fraction (5/7)}of the region of interest, followed by coupling of B. It will berecognized that mask 292 may in fact be the same mask as 291 buttranslated across the substrate. Accordingly, regions indicated bydashed line 290, may also be exposed to light in this step, as well asin later steps. Thereafter, subsequent masking steps expose {fraction(4/7)}, {fraction (3/7)}, {fraction (2/7)}, and {fraction (1/7)} of thearea of interest on the substrate, each mask being used to couple adifferent monomer (C, D, E, F) to the substrate. The resulting substrateis schematically illustrated in the bottom portion of FIG. 1A along withthe resulting polymer sequences thereon. Again, the composition of thesequences on the substrate is given by the vertical column such as“ABCDEF.” As seen, 1-to 6-membered truncated portions of the targetABCDEFG are formed.

[0110]FIG. 1B illustrates the next series of masking steps. As shown inFIG. 1B, the same mask 293 is used for each masking step, but the maskis translated with respect to the substrate in each step. In each step,the mask illuminates a portion of each of the “stripes” of polymersformed in FIG. 1A, and in each step a different one of the monomers in abasis set is coupled to the substrate. The mask is then translateddownwards for irradiation of the substrate and coupling of the nextmonomer. In the first step, the mask exposes the top {fraction (1/20)}of each “stripe” of polymers shown in FIG. 1A, and monomer X₁ is coupledto this region. In the second step, the mask is translated downwards andX₂ is coupled, etc. The resulting substrate is shown in the bottomportion of FIG. 1B. An additional stripe 295 is formed adjacent theregion addressed in FIG. 1A, this region containing a series ofsubregions, each containing one of the 20 monomers in this particularbasis set.

[0111] Accordingly, after the steps shown in FIG. 1B, the substratecontains the following polymer sequences on the surface thereof (columnsagain indicating the sequences formed on the substrate): X X F X E E X DD D X C C C C X B B B B B X A A A A A A

[0112] where X indicates that an individual region contains one of eachof the monomers in the basis set. Accordingly, for example, each of the20 dimers AX are generated when the basis set is the 20 natural L-aminoacids typically found in proteins. The 20 trimers of ABX, 4-mers ofABCX, etc., are also formed at predefined regions on the substrate.

[0113] Thereafter, as shown in FIG. 1C, the process continues,optionally using the same mask(s) used in FIG. 1A. The masks differ onlyin that they have been translated with respect to the substrate. In step27, monomer B is added to the substrate using the mask that illuminatesonly the right {fraction (1/7)} of the region of the substrate ofinterest. In step 28, the right {fraction (2/7)} of the substrate isexposed and monomer C is coupled, etc. As shown in the bottom portion ofFIG. 1C, the process results in the generation of all possible polymersbased on the polymer ABCDEFG, wherein each monomer position issystematically substituted with all possible monomers from a basis set.

[0114] A number of variations of the above technique will be useful insome applications. For example, in some embodiments the process isvaried slightly to form disubstitutions of a lead polymer in which thesubstitutions are in adjacent locations in the polymer. Such arrays areformed by one of a variety of techniques, but one simple techniqueprovides for each of the masks illustrated in steps 7-26 of FIG. 1 tooverlap the previous mask by some fraction, e.g., ⅓, of its height.According to such embodiments, the following array of polymers would begenerated, in addition to the previous array of 134: G G G G G G X F F FF F X X E E E E X X F D D D X X E E B X X C C C C X X B B B B B X A A AA A A

[0115] The scheme may be expanded to produce tri-substituted,tetra-substituted, etc. molecules. Accordingly, the present inventionprovides a method of forming all molecules in which at least onelocation in the polymer is systematically substituted with all possiblemonomers from a basis set.

[0116] According to a preferred aspect of the invention, masks areformed and reused to minimize the number of masks used in the process.FIG. 2 illustrates how masks may be designed in this manner. Forsimplicity, a 5-monomer synthesis is illustrated. The masks areillustrated from “above” in FIG. 2, with the cross hatching indicatinglight-transmissive regions. The resulting substrate is shown in thebottom portion of FIG. 2, with the region of primary interest formono-substitutions indicated by the arrows.

[0117]FIG. 2 illustrates how to generate and systematically substituteall of the 5-mers contained in a 6-mer lead. A limited set of thepossible 2-, 3-, and 4-mers is also synthesized. A 4-pattern mask wasused. To make the 6-mers in a 7-mer kernel, a 5-pattern mask is used. Tomake the 7-mers in an 8-mer kernel, a 6-pattern mask is used, etc. Tomake all the 7-mers in a 12-mer kernel, a 6-pattern mask is still allthat is needed. As a “bonus,” all of the truncation sequences aregenerated with this strategy of letting the masks extend beyond the“desired” regions.

[0118] For example, FIG. 2 illustrates that in a 6-monomer sequence the1- to 5-position substituted polymers are formed in the primary regionsof interest (indicated by arrows), the 2- to 6-position substitutedpolymers are formed in region 403, the 3- to 6-position substitutedpolymers are formed in region 404, the 4- to 6-position substitutedpolymers in region 405, etc. The 1- to 4-position substituted polymersare formed in regions 406, the 1- to 3-position substituted polymers inregion 407, etc.

[0119] In some embodiments, the substrate is only as large as the regionindicated by arrows. It will often be desirable, however, to synthesizeall of the molecules illustrated in FIG. 2 since the deletion sequencesand others found outside of the region delineated by arrows will oftenprovide additional valuable binding information.

[0120] As shown, a single mask 401 is used for all of steps 1-5, whileanother mask 402 is used for steps 6-11. The same mask 401 is used forsteps 12-16, but the mask is rotated preferably 180 degrees with respectto the substrate. The light tranmissive regions of the mask 401 extendthe full length (“Y”) of the area of interest in the y-direction. Asshown, in step 1, the mask is placed above the substrate, the substrateis exposed, and the A monomer is coupled to the substrate in selectedportions of the substrate corresponding at least to the region 401 a.Monomer A may also be placed at other locations on the substrate atpositions corresponding to mask regions 401 b, 401 c, and 401 d.Alternatively, these regions of the mask may simply illuminate regionsthat are off of the substrate or otherwise not of interest. If theseregions correspond to regions of the substrate, various truncatedanalogs of the sequences will be formed.

[0121] Thereafter, as shown in step 2, the same mask is utilized, but itis translated to the right. The substrate is exposed by the mask, andthe monomer B is then coupled to the substrate. Thereafter, couplingsteps 3, 4, and 5 are conducted to couple monomers C, D and E,respectively. These steps also use the same mask translated to the rightin the manner shown.

[0122] Thereafter, mask 402 is used for the “X” coupling steps. The mask402 contains a single stripe that extends the full length (“X”) of thearea of interest in the x-direction. Mask 402 is preferrably a single,linear stripe that will normally be of width Y divided by the number ofmonomers in the substitution basis set. For example, in the case of 6amino acids as a basis set for the monosubstitution of peptides, thestripe will have a width of Y/6. The mask is repeatedly used to coupleeach of the monomers in the basis set of monomers and is translateddownwards (or upwards) after each coupling step. For example, the maskmay be placed at the top of the region of interest for the firstcoupling step, followed by translation downwards by ⅙ of the Y dimensionfor each successive coupling step when 6 monomers are to be substitutedinto the target. FIG. 2 shows only 2 mask steps for simplicity, but agreater number will normally be used.

[0123] Thereafter the mask 401 is again utilized for the remainingcoupling steps. As shown, the mask 401 is rotated, preferably 180degrees, for the following coupling steps. The succeeding coupling steps12-16 are used to couple monomers B-F, respectively. The resultingsubstrate is shown in “cross section” in the bottom portion of FIG. 2.Again, the primary area of interest is designated by arrows and may bethe only region used for synthesis on the substrate. The truncatedsubstitutions outside of this region will also provide valuableinformation, however.

[0124] One extension of this method provides for the synthesis of allthe possible double substitutions of a kernel sequence. For a kernelsequence 7 residues long, there are 8400 peptides that make up allpossible disubstitutions of 20 amino acids, not considering replication(8380 unique). These peptides can be synthesized in 55 steps with 17masks. The synthesized sequences are shown below: G G G G G X G G G G XG G G X G G X G X X F F F F X F F F F X F F F X F F X F X F X E E E X EE E E X E E E X E E X E E X X E D D X D D D D X D D D X D D D X X X D DD C X C C C C X C C C C X X X X C C C C C C X B B B B B X X X X X B B BB B B B B B B X X X X X X A A A A A A A A A A A A A A A

[0125] The systematic substitution of three or more positions of thekernel sequence is also easily derived. The optimum polymer identifiedfrom the above strategy can then serve as the new kernel sequence infurther iterations of this process. The present method may be used forany desired systematic substitution set, such as all 8-mers in a 12-merkernel, substitution in cyclic polymers, and the like. This methodprovides a powerful technique for the optimization of ligands that bindto a molecular recognition element.

[0126] B. Cyclic Polymer Mapping

[0127] Copending application Ser. No. 07/796,727 (Entitled “PolymerReversal on Solid Surfaces”). incorporated herein by reference for allpurposes, discloses a method for forming cyclic polymers on a solidsurface. According to one aspect of the present invention, improvedstrategies for forming systematically varied cyclic polymers areprovided.

[0128] In the discussion below, “P” refers to a protecting group, and X,Y, and Z refer to the various reactive sites on a tether molecule T. A,B, C, D, E, and F refer to various monomers or groups of monomers. Tosynthesize a cyclic polymer according to one aspect of the inventionherein, the process is conducted on a substrate. A tether molecule T iscoupled to a surface of the substrate. T may be one of the monomers inthe polymer, such as glutamic acid in the case of amino acids. Otherexamples of amino acid tether molecules include, but are not limited to,serine, threonine, cysteine, aspartic acid, glutamic acid, tyrosine,4-hydroxyproline, homocysteine, cysteinesulfinic acid, homoserine,ornithine, and the like. The tether molecule includes one or morereactive sites such as a reactive site Z which is used to couple thetether to the substrate. The tether also includes a reactive site Xhaving a protecting group P₂ thereon. The tether molecule furtherincludes a reactive site Y with a protecting group P₁ thereon.

[0129] In a first step, a polymer synthesis is carried out on thereactive site Y. According to some embodiments, conventional polymersynthesis techniques are utilized such as those described in Atherton etal., previously incorporated herein by reference for all purposes. Awide variety of techniques may be used in alternative embodiments. Forexample, according to one embodiment, a variety of polymers withdifferent monomer sequences are synthesized on the substrate. Suchtechniques may involve the sequential addition of monomers or groups ofmonomers on the growing polymer chain, each monomer of which may alsohave a reactive site protected by a protecting group.

[0130] A variety of such methods are available for synthesizingdifferent polymers on a surface. For example, Geysen et al., “Strategiesfor Epitope Analysis Using Peptide Synthesis,” J. Imm. Meth., (1987)102:259-274, incorporated herein by reference for all purposes,describes one commonly used technique for synthesizing differentpeptides using a “pin” technique. Other techniques include those ofHoughten et al., Nature (1991) 354:84-86, incorporated herein byreference. In some embodiments, advanced techniques for synthesizingpolymer arrays are utilized, such as those described in copendingapplication Ser. No. 07/796,243, or light-directed,spatially-addressable techniques disclosed in Pirrung et al., U.S. Pat.No. 5,143,854; U.S. application Ser. No. 07/624,120; and Fodor et al.,“Light-Directed Spatially-Addressable Parallel Chemical Synthesis,”Science (1991) 251:767-773, all incorporated herein by reference for allpurposes, such techniques being referred to herein for purposes ofbrevity as VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis)techniques.

[0131] During polymer synthesis, the activator used to remove P₁ (ifany) on the Y reactive site, and on reactive sites of the growingpolymer chain, should be different than the activator used to remove theX protecting group P₂, Merely by way of example, the activator used toremove P₂ may be a first chemical reagent, while the activator used toremove the protecting group P₁, may be a second, different chemicalreagent such as acid or base. By way of further example, the activatorused to remove one of the protecting groups may be light, while theactivator used to remove the other protecting group may be a chemicalreagent, or both activators may be light, but of different wavelengths.Of course, other combinations will be readily apparent to those of skillin the art on review of this disclosure.

[0132] By virtue of proper protecting group selection and exposure toonly the P₁ activator, the reactive site X is protected during polymersynthesis and does not take part in the initial portion of the process.Also, the reactive site Y remains bound to the monomer A. The synthesisstep of the process, which will frequently include many substeps ofdeprotection/coupling, results in a polymer of a desired length, such asABCD . . . F. A polymer with 5 or more monomers is used by way ofexample, but fewer (or more) monomers will be utilized according to someembodiments.

[0133] In a next step of the process, the protecting group P₂ on the Xreactive site is removed. In addition, the reactive site on the lastmonomer F is rendered active, if necessary. The reactive site on theselected monomer will then react with the reactive site X, forming acyclic polymer. In a preferred embodiment for peptide synthesis, theprotecting group P₂ is removed with light.

[0134] Choice of the various protecting groups will generally bedictated by the type of polymer which is to be synthesized and thedesired synthesis technique. Therefore, for example, oligonucleotideswill often have different protecting groups than will peptides,oligosaccharides, and the like. In addition, conventional solid-phasesynthesis techniques without the use of photoremovable protecting groupswill utilize different protecting groups than VLSIPS™ light-directedsynthesis techniques. Specific examples of protecting groups arediscussed in detail below. Table 1 summarizes the various protectinggroups used according to most preferred embodiments of the invention.TABLE 1 Preferred Protecing Group Selections P₁/ P₂/ Synthesis ActivatorActivator Standard FMOC/ NVOC Peptide Base or other photochemical/ baseor light Standard BOC/ NVOC Peptide Acid or other photochemical/ base orlight Standard DMT/ NVOC Nucleotide Mild Acid or other photochemical/light VLSIPS ™ NVOC (or FMOC, allyl, Peptide other photo- silyl, orother chemical base sens./ protecting base groups)/ Light VLSIPS ™ NV orDMT Nucleotide NVOC/ or other Light acid sens./ acid

[0135] One technique of standard Merrifield peptide synthesis employsfluorenylmethyloxycarbonyl (FMOC) on the growing end (amino terminus) ofthe polymer and one or more of a variety of side chain protectinggroups. According to preferred embodiments herein, such techniquesgenerally utilize mild base treatment to remove the FMOC (P₁) forpeptides and strong acid (up to 100% TFA) for both removal of the sidechain protecting groups and cleavage of the tether/polymer bond. Base orlight is used to remove the protecting group P₂, which may be, forexample, NVOC.

[0136] One embodiment of the invention utilizes a group P₁ which isremovable with a first wavelength of light and a second photocleavablegroup P₂ which requires a different wavelength for deprotection of X.Preferably such groups utilize wavelengths >300 nm to avoid conflictingwith protecting groups in use during polymer synthesis and to avoiddamage to sensitive amino acids. Alternatively, some embodiments employa base-, paladium- or fluoride-sensitive protecting group. Other suchmaterials include FMOC, β cyanoethyl, t-butyldiphenylsilyl, allyl andothers apparent to those of skill in the art.

[0137] Cyclic polymers immobilized on a surface present uniqueopportunities for biological activity screening. For example, with acyclic polymer it is desirable to vary not only the monomers in thepolymer, but also the point at which the cyclic polymer attaches to thesubstrate and, therefore, the region of the polymer available forbinding. Further, depending on the building blocks of the polymer, oneor more of the building blocks may not be amenable to attachment to asubstrate.

[0138]FIG. 3 illustrates this aspect of the invention. In the particularembodiment shown in FIG. 3, a cyclic polymer made from 8 monomers isillustrated. As shown therein, it is desirable to synthesize the polymerso that it is attached to the substrate at different positions in thering. For example, the left-most molecule in FIG. 3 is attached to thesubstrate with monomer 1, the second polymer is synthesized such that itis attached to the substrate via monomer 2, and the third polymer isattached to the substrate via monomer 3. In the most general case, it isdesirable to synthesize an array of cyclic polymers in which a polymeris attached to the substrate via each of the monomer positions. Forexample, with reference to FIG. 3, it is desirable to attach the8-monomer polymer via each of the 8 monomers therein. The site availablefor recognition will be slightly different for the polymer for eachattachment position, because the polymer is presented in a “rotated”position on the various regions of the substrate.

[0139] Again, while the invention is illustrated with regard to cyclicpolymers with about 8 monomers, a wide range of polymers may be utilizedin conjunction with the invention without departing from the scopethereof. For example, when the polymers are peptides, the polymermolecules will typically contain between about 4 and 10 monomers, oftenbetween about 6 and 8 monomers.

[0140] Difficulties arise, however, in the attachment of certainmonomers to the substrate. For example, in the case of amino acids,certain amino acids do not have side groups that are amenable toattachment to a solid substrate. Accordingly, in one embodiment of theinvention, an array of cyclic polymers is synthesized in which themonomer used for attachment to the substrate is readily attachable tothe substrate, such as glutamic acid. Like the first embodiment, thepolymers have a common monomer sequence. However, the monomer used forcoupling in all of the sites, according to this embodiment, is a commonmonomer that is easily coupled to the substrate.

[0141] The substrate attachable monomer may either be substituted intothe native polymer sequence at various locations by alternativelydeleting one member of the polymer chain or by inserting the substrateattachable monomer into the native polymer sequence at differentlocations. FIGS. 4 and 5 illustrate these alternatives, with FIG. 4illustrating a substitution strategy (in which a selected tethermolecule is substituted into the native ring at the coupling site), andFIG. 5 illustrating an insertion strategy (in which a selected tethermolecule is added to the native ring at the coupling site).

[0142] As shown in FIG. 4, substrate attachable monomer “A,” referred toelsewhere herein as a tether, is coupled to the substrate and links thecyclic polymer to the substrate. In preferred embodiments, A substitutesfor the monomer that would otherwise be in the position of the polymerthat is coupled to the substrate. This substitution preserves theoriginal ring size. For example, in the cyclic polymer molecule in theleft portion of FIG. 4, the monomer A has been substituted for themonomer 1. The monomer A has been substituted for the monomer 2 in thepolymer in the second portion of FIG. 4. The monomer A has beensubstituted for the monomer 3 in the polymer in the next polymer of FIG.4.

[0143] In most embodiments, it will be desirable to synthesize an arrayof cyclic polymers in which the polymers are coupled to the substrate ateach monomer position in the polymer. For example, in the case of an8-mer, there will be 8 different attachment positions for the polymer.Often, it will be desirable to synthesize arrays of polymerssimultaneously in which not only is the attachment position varied foran individual monomer, but different polymer molecules are formed on thesubstrate. For example, in the case of a cyclic pentapeptide with allpossible combinations of natural amino acid monomers and all possibleattachment positions, it may be desirable to synthesize all 800,000combinations of sequence and attachment locations on one or moresubstrates.

[0144] According to a preferred aspect of the invention, a single maskmay be used to form cyclic polymers with varying points of attachment ona substrate. FIGS. 6A to 6E illustrate one preferred masking strategy,using a 7-monomer cyclic polymer as an example. Formation of the rotatedcyclic polymers on the same substrate with formation of polymers havingdifferent monomers at the various positions of the polymers represents apreferred embodiment of this invention. Only those steps relevant to theformation of rotated cyclic polymers are outlined below for the purposeof simplicity in the illustration.

[0145] As shown in FIG. 6A, the process begins with the illumination andcoupling of molecule A to the surface in a region of interest 281.Molecule A may be attached directly or indirectly (via linkers) to thesolid substrate. Molecule A is provided with terminal protecting groupP₁ and side protecting group P₂ if necessary. P₁ and P₂ are removableunder different conditions. For example, in a preferred embodiment ofthe invention Pi is removable upon exposure to light and P₂ is removableupon exposure to, for example, acid or base. Details of various tethermolecules A and protecting groups are included in copending applicationSer. No. 07/796,727, previously incorporated herein by reference.

[0146] Thereafter, as shown in FIG. 6B, the mask is translated so as toexpose only the left-most portion of the region of interest, and monomer1 is coupled to the substrate. Monomer 1 also has a photoprotectinggroup on a terminus thereof. As shown, monomer 1 may also be coupled onareas of the substrate that are not in the region of interest, or theregion to the left of the region of interest may be off of the edge ofthe substrate. The left and right portions of the substrate will,accordingly, be ignored in the illustrations below.

[0147] Thereafter, the process continues as illustrated in FIG. 6C, inwhich monomer 2 is coupled to the growing polymer chain using the samemask, again translated by one position. Processing continues withsuccessive exposures to light using the translated mask, followed bycoupling of the appropriate monomers, resulting in the substrate shownin FIG. 6D. Thereafter, the photoprotecting groups are removed from theterminus of all of the terminal monomers.

[0148] Then, the side chain protecting groups are removed from each ofthe tether molecules A, followed by coupling of the terminal monomers tothe formerly protected side chain groups, in accordance with theteachings of Ser. No. 07/796,727. Accordingly, an array of polymers isproduced as shown in FIG. 6E that contains spatially addressable regionscontaining the cyclic 7 member polymer, each coupled at a differentposition in the polymer via a common tether molecule.

[0149] In some embodiments it may be desirable to identify a targetcyclic polymer, and synthesize an array of cyclic polymers in which notonly is the polymer rotated, but also in which the monomers aresystematically substituted with various monomers from a basis set. Forexample, it may be desirable to synthesize all polymers in which the 6thbuilding block is systematically substituted with 20 L-amino acids.

[0150] In accordance with this aspect of the invention, an “X” mask isincluded in the synthesis strategy, similar to the method describedabove for linear polymers. The X mask is used to couple each of themonomers from a basis set in a selected position of the cyclic polymer.Accordingly, the resulting array in preferred embodiments contains allof the polymers in which one position is systematically varied. Inaddition, for each polymer, the polymer is coupled via each position inthe monomer to the substrate.

[0151] For purposes of illustration, a cyclic polymer of 7 residues isshown. FIGS. 7A to 7C illustrate one preferred masking strategy forforming such cyclic polymer arrays. The particular embodiment shown inFIG. 7 illustrates substitution of monomers at the “6” position. Asshown in FIG. 7A, processing of the substrate is initially the same asthat shown in FIG. 6, in which the A tether as well as monomers 1-5 arecoupled to the substrate. As shown in FIG. 7B (top view), the substrateis then processed with an “X” mask. The X mask is used to couple thevarious monomers from a basis set to the substrate. The X mask may bethe same for coupling of each member of the basis set and is simplytranslated for the coupling. For example, in the first exposure, the Xmask is arranged in the form of a horizontal rectangle at the top of theregion of interest on the substrate, and the first monomer from thebasis set (X₁) is coupled in this region. The X mask traverses each ofthe regions shown in FIG. 7A. The mask is then translated downwards, andthe second monomer in the basis set (X₂) is coupled in this region,again with the mask traversing each of the regions formed up to the stepshown in FIG. 7A. The mask is translated successively downwards until,for example, all 20 monomers in the basis set are coupled at variousregions of the substrate, as indicated in FIG. 7B.

[0152] Thereafter, the original mask is again utilized. The mask isfirst used to couple monomer 7 to the right ⅚ of the substrate shown inFIG. 7B. The mask is then translated and used to couple monomer 1 to theright {fraction (4/6)} of the substrate shown in FIG. 7B. The mask isthen used to couple monomer 2 to the right {fraction (3/6)} of thesubstrate shown in FIG. 7B. Successive couplings are conducted withmonomers 3, 4, and 5. The sequences of monomers on the resultingsubstrate are illustrated below, where X indicates that a region of thesubstrate contains polymers with each of the basis set of monomersselected for insertion at the 6th position of the polymer. The polymersequences are obtained by examination of the columns of the illustrationbelow: X 7 1 2 3 4 5 X 7 1 2 3 4 5 X 7 1 2 3 4 5 X 7 1 2 3 4 5 X 7 1 2 34 5 X A A A A A A

[0153] Thereafter, the polymers are cyclized, resulting in an array ofpolymers in which every member of the basis set is inserted at the sixthposition of the polymers, and in which each polymer thus synthesized iscoupled to the substrate at each rotational position in the polymer, allat spatially addressable regions. A portion of the resulting array isillustrated in FIG. 7C.

[0154] To vary every building block at every position systematicallyrequires a different set of masks. For the above 7-numbered cyclicpeptide, the length of the synthesis region is divided into 36 equalunits, while the width of the synthesis region is divided into “x” units(typically x=20 for peptides with “natural” amino acids). The resultingpolymer library will be as follows, again with columns indicating theresulting polymer sequence and “X” indicating that polymers with allmembers of a basis set substituted at that position are formed:66666X11111X22222X33333X44444X55555X5555X56666X61111X12222X23333X34444X4444X44555X55666X66111X11222X22333X3333X33344X44455X55566X66611X11122X2222X22223X33334X44445X55556X66661X1111X11111X22222X33333X44444X55555X66666AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0155] In synthesizing the above polymers, the order of coupling willbe: A1234561234X2345612345. FIG. 8 illustrates the cyclic polymersresulting from the synthesis. FIG. 9 illustrates a convenient mask usedin the synthesis.

[0156] This strategy differs slightly from those discussed above in thatthe synthesis produces 7-membered rings in which the tether polymer isadded to 6-membered kernel sequence. To mimic the structure of theactual polymer more closely, it is desirable in some embodiments to usea tether molecule that is shorter than the monomer molecules so as tomaintain the native length of the molecule. For example, in the case ofpeptides, a disulfide molecule serving as the tether will be beneficial.Examples of such cyclic peptides with a disulfide linkage are common inthe literature, i.e., oxytocin and vasopressin. The 6 amino acids andthe disulfide linkage will produce a 20-membered ring. By comparison, acyclic hexamer of a peptide is an 18-membered ring, while a cyclicpeptide heptamer will be a 21-membered ring. Accordingly, when thedisulfide molecule is inserted into the cyclic polymer, the tether willproduce polymers that more correctly mimic the native polymer. Otheruseful tether molecules include glutamic acid, as illustrated in FIGS.10A and 10B.

[0157] The following series of polymers can be made with the same masks(one to lay down “A”, two with 180° symmetry to assemble the polymer,and one “X” mask). This method produces all single substitutionspossible while maintaining the rest of the cycle constant. One couldalso use the method to make double and triple substitutions.77777X11111X22222X33333X44444X55555X66666X6666X67777X71111X12222X23333X34444X45555X5555X55666X66777X77111X11222X22333X33444X4444X44455X55566X66677X77711X11122X22233X3333X33334X44445X55556X66667X77771X11112X2222X22222X33333X44444X55555X66666X77777X11111AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0158] In the above set of polymers A is the molecule used to couple thepolymer to the substrate, 1-7 are amino acids or other monomers in thekernel sequence, and X are the substitutions from the basis set ofmonomers. One way to assemble this array is: A23456712345X34567123456.The masks that would be utilized before the X coupling are shown in FIG.11A, and the masks used after the X coupling are shown in FIG. 11B.

[0159] III. Data Collection

[0160] A. CCD Data Collection System

[0161] Although confocal detection systems are typically used for datacollection, according to one embodiment a high resolution CCD camerasystem is utilized for data collection. The camera allows for thedigitization of images with a resolution of, e.g., 1300 by 1024 pixels.A dynamic range of >60 dB can be obtained if the sensor is calibratedwith respect to dark current and gain. Cooling the camera to 248° K.lowers the dark current to a tolerable level even when prolongedexposure times (several minutes) are needed.

[0162] According to one embodiment, a 100 W Hg-Arc lamp is used as alight source. The infrared components of the light are blocked using aheat-absorbing filter. A second filter is used to select the excitationwavelength using an optional ground glass plate filter. Best results areachieved by illuminating the sample with UV. Although the optimumexcitation wavelength for FITC lies at 490 nm, excitation in the UVrange shows better results than with a 490 nm IF filter, because the Hglamp provides more optical energy in the UV band. The sample isilluminated at an angle of 450 with respect to the light beam and theCCD camera. This ensures that no direct light path exists between thelamp and the sensor and therefore reduces background radiation.

[0163] The CCD camera is mounted at the back of a Hasselblad 500 C/Mcamera system with a lens and IR filter. An automatic bellow and astandard 80 mm (f 2.8) lens are used. This system results in an imagingscale which is selectable between 1:3 and 1:0.8. The sample plus thefilters are housed in a case to prevent light from the surroundings fromentering the optical path. Digitized image data are transmitted to a 386PC, where the images can be viewed on a high resolution display.

[0164] Longer integration times (up to 2 minutes) yield better S/Nratios. One problem is the fluorescence (and light diffraction) of dustparticles on the surface of the VLSIPS™ chip. Dust particles deliver asignal about 10 times higher than the FITC fluorescence. Therefore, theintegration time can be increased only as long as the dust particles donot cause an overflow (blooming) of the sensor. However, since an areaof about 50×50 pixels can be averaged in the digitized image for aquantitative assessment of the fluorescence, the measurement accuracy issufficient with integration times between 15 and 30 seconds.

[0165] If the acquisition time needs to be lower than the 15-20 secondsmentioned above, a different light source should be selected. An argon(λ=488 nm) laser is optimal. The disadvantage of using a laser is theconsiderably higher technological expense, and the possibility ofbleaching if the optical energy is raised too much. Bleaching could notbe observed while using the Hg lamp (even with integration times ofseveral minutes). Using a lens with a lower focal length (e.g., astandard 50 mm / f 1.4 lens) would improve the overall efficiency of thesystem.

[0166] The optimized optical system could allow for measurement timesbelow 10 seconds using a Hg lamp. When using a laser, image acquisitionwill take less than 1 second. The system should be operated in adust-free environment (such as a lateral flow workbench) to reduceerrors generated by dust particles.

[0167] B. Trapping Low Affinity Interactions

[0168] According to one embodiment, the invention provides a methodologyfor chemically trapping-low affinity interactions between receptors andimmobilized ligands. Monovalent receptors with K_(d)'s greater than 100nM may not bind with sufficient affinity to an immobilized ligand tosurvive subsequent washing and imaging steps for later detection. Thus,while high concentrations (approximately near the K_(d) of the peptidelead) of receptor should bind to some epitopes on, for example, aVLSIPS™ chip, this information may be lost during subsequent processing.Accordingly, cross-linkers are used according to one embodiment of theinvention. The cross-linkers are designed to be specific for thereceptor-ligand complex while having relatively no specificity for freereceptor. Accordingly, it is possible not only to trap covalently thereceptor, but also recover the excess receptor in unmodified form.

[0169] To accomplish this the invention provides:

[0170] 1. A residue or “handle” common to all ligands on the solidsupport.

[0171] 2. Heterobifunctional crosslinking agents in which one of thefunctionalities alkylates the “handle” kinetically much faster than itwould alkylate either the receptor or the immobilized ligand.

[0172] 3. The second functionality of the heterobifunctional crosslinkeralkylating the immobilized receptor kinetically much faster than itwould alkylate either the free receptor or the immobilized ligand.

[0173] According to one embodiment, the substrate is “doped” byreplacing a small amount of the NVOC-aminocaproic acid reagent on thesurface of the substrate with a small quantity of t-Boc-mercaptocaproicacid. The mercaptan would be blocked during all of subsequent peptidesynthesis steps, but would deprotect upon exposure to acid. The surfacewould then be treated with receptor solution and the free sulfhydrylgroup acts as an outstanding nucleophile that is alkylatedinstantaneously upon treatment with a variety of commercially availableheterobifunctional crosslinkers. In a second crosslinking step,alkylation of bound receptor is facile, because the proximity of theprotein makes this reaction pseudo first-order. Nonspecific alkylationof free receptor would be a second-order process and thereforedisfavored over specific alkylation. The excess receptor would then beremoved and recovered by fast-desalting chromatography.

[0174] Because the receptor is now covalently bound to the substrate,subsequent processsing will not remove the receptor enabling detectionof event those receptors with a high Kd.

[0175] C. Fluorescence Energy-Transfer Substrate Assays

[0176] A different application of the present invention tests forcatalytic cleavage of various polymer sequences by an enzyme or othercatalyst. For example, aspartyl proteases such as renin, HIV proteases,elastase, collagenase and some cathepsins can be tested against an arrayof peptides. According to this aspect of the invention, a variety ofpeptide sequences are synthesized on a solid substrate by theprotection-deprotection strategy outlined above. The resulting array isprobed with an enzyme which might cleave one or more peptide elements ofthe array resulting in a detectable chain.

[0177] In one embodiment, the peptides to be tested have a fluorescencedonor group such as 1-aminobenzoic acid (anthranilic acid or ABZ) oraminomethylcoumarin (AMC) located at one position on the peptide and afluorescence quencher group such as lucifer yellow, methyl red ornitrobenzo-2-oxo-1,3-diazole (NBD) at a different position near thedistal end of the peptide. Note, that some “donor” groups can also serveas “quencher” groups, depending on the relative excitation and emissionfrequencies of the particular pair selected. The intramolecularresonance energy transfer from the fluorescence donor molecule to thequencher will quench the fluorescence of the donor molecule. Uponcleavage, however, the quencher is separated from the donor group,leaving behind a fluorescent fragment. A scan of the surface with anepifluorescence microscope, for example, will show bright regions wherethe peptide has been cleaved. FIG. 12A shows a tripeptide having adonor-quencher pair on a substrate. The fluorescence donor molecule,1-aminobenzoic acid (ABZ), is coupled to the ε-amino group of lysine(Lys) on the P′ side of the substrate. The donor molecule could, ofcourse, be attached to the α-amine group. A fluorescence quencher, NBDcaproic acid, is coupled to the P side of the substrate molecule. Uponcleavage by a protease, as shown in FIG. 12B, the quencher is releasedleaving the fluorescent fragment still bound to the solid substrate fordetection.

[0178]FIGS. 13A to 13H illustrate various alternative donor/quencherpairs which are used according to alternative embodiments of theinvention.

[0179] IV. Examples

[0180] A. Example

[0181] The binding interactions of a monoclonal antibody D32.39, whichwas raised against the opioid peptide dynorphin B (YGGFLRRQFKVVT), wereexplored. The binding of antibody D32.39 to dynorphin B was previouslyshown to be directed to the carboxyl terminus. Initially, the experimentaddressed the minimum peptide size required for binding and the locationof the antibody binding epitope within the full length peptide. A binarymasking strategy was used to generate an array of peptides of all thelinear sequences contained within the terminal ten residues(FLRRQFKVVT). The ten-step synthesis was configured to yield fourreplicates of the 1024 possible compounds (i.e., 4096 distinct polymersynthesis regions), of which 1023 were peptides ranging in length fromone to ten residues in length, with a mean length of five. This arrayincludes every possible truncation and deletion sequence, as well asmultiple deletions, contained within these ten residues, whilepreserving the linear order of amino acids. Due to the redundancy inamino acids (two valines, two arginines, and two phenylalanines), 560unique sequences were generated.

[0182] After synthesis of the array, the terminal NVOC protecting groupswere removed, the terminal amines were acetylated, and the side chainprotecting groups were cleaved under standard conditions. Non-specificprotein binding to the surface was blocked with 1% BSA/PBS/0.05% Tween20. The array was incubated with the D32.39 antibody at a concentrationof 10 μg/ml for 2 hours, followed by reaction with 10 μg/ml of FITCconjugated anti-mounse antibody (Sigma) for 2 hours at 20° C. Followingextensive washing with buffer, the array was scanned in a confocalfluorescence microscope with 488 nm excitation from an argon ion laser(Spectra-Physics). The resultant fluorescence image containing theintensity versus positional data for the array of peptides wasnormalized from 0 to 100% relative intensity by subtracting outbackground (the region within the array containing only the linkermolecule) and using the region of highest intensity (FRQFKVVT) as 100%relative intensity. The replicates were averaged, and the data sortedaccording to desired sequences. The entire screening process, includingpeptide synthesis and data acquisition and workup, required three daysto complete.

[0183] A survey of the data obtained from the ten possible singledeletion peptides affords a preliminary estimate of the regions withinthe kernel sequence responsible for binding to the antibody. Thenormalized fluorescence intensity for each of these deletion sequencesare is in FIG. 13. The full length peptide exhibited about 100% relativesignal as anticipated, indicating that the epitope lay within the 10-mersequence. Deletions in the kernel sequence near the amino terminus hadlittle effect on the observed signal (compare F, L, R, R deletions),while deletions near the carboxyl terminus (compare F, K, T deletions)had a more pronounced effect. The antibody shows intermediate relativebinding to both the valine and glutamine deletions.

[0184] An analysis of the terminal truncated peptides generated in thearray allows one to draw conclusions regarding the size and location ofthe epitope contained in the kernel sequence. The normalizedfluorescence signals obtained for the terminal truncated peptides areshown in FIG. 14. The data reveals that truncations from the aminoterminus are tolerated until loss of the second arginine residue,indicating the importance of the arginine residue to antibodyrecognition. Truncations from the carboxyl terminus are not tolerated aswell, and the initial truncation of the threonine residue results in alarge decrease in the observed fluorescence signal. The combination ofthese two observations predicts that the epitope lies between, andincludes both, the arginine and threonine residues, or hence RQFKVVT.

[0185] Examination of the signals observed for all the possibletruncated sequences in Table 2 contained within the kernel sequenceaffords the highest degree of confidence in assigning the size andposition of the epitope. Peptides shorter than seven residues wereobserved to show diminished binding to the antibody. Binding of theD32.39 antibody to the immobilized peptides exhibits a strong biastowards the RQFKVVT sequence. TABLE 2 Sequence and Relative FluorescenceIntensities of the Truncated Dynorphin Peptides Sequence NormalizedIntensity (%)^(a) FLRRQFKVVT 98 ± 1 LRRQFKVVT  87 ± 13 FLRRQFKVV  37 ±15 RRQFKVVT 99 ± 1 LRRQFKVV  44 ± 12 FLRRQFKV 14 ± 3 RQFKVVT 99 ± 1RRQFKVV  58 ± 10 LRRQFKV 16 ± 3 FLRRQFK 10 ± 3 QFKVVT 28 ± 3 RQFKVV  54± 11 RRQFKV 16 ± 3 LRRQFK 12 ± 3 FLRRQF  8 ± 3 FKVVT 13 ± 4 QFKVV 17 ± 2RQFKV 16 ± 4 RRQFK 12 ± 3 LRRQF 10 ± 4 FLRRQ  8 ± 3 KVVT 11 ± 2 FKVV 10± 2 QFKV 11 ± 2 RQFK 13 ± 5 RRQF 11 ± 5 LRRQ 10 ± 4 FLRR  6 ± 3

[0186] To confirm the interpretation of the observed fluorescencesignals, peptides synthesized on a conventional solid phase peptidesynthesizer were tested. The IC₅₀ values for the competition of freepeptide against radiolabeled dynorphin B peptide were determined and aretabulated in Table 3. There is a striking correlation between the rankordering of the relative fluorescence intensity and the solution IC₅₀values. Although the antibody appears to require the presence of thethreonine residue at the carboxyl terminus, it shows little preferencefor the free carboxamide versus the free acid. TABLE 3 Solution BindingData for the Dynorphin Peptides to the D32.39 Antibody SequenceIC_(50 (μM)) ^(a) Normalized Intensity (%)^(b) YGGFLRRQFKVVT-OH 0.0057nd^(c) Ac-FLRRQFKVVT-OH nd 98 Ac-RRQFKVVT-OH 0.0039 99 Ac-RQFKVVT-OH0.011 99 Ac-RQFKVVT-NH₂ 0.0073 — Ac-QFKVVT-OH 3.2 28 Ac-FKVVT-OH 77.0 13

[0187] The relative fluorescence intensity observed for biologicalrecognition of an antibody to an array of immobilized peptides dependson several factors. Of primary importance is the multivalent interactionbetween the antibody and the surface due to the presence of two antibodycombining sites in an IgG molecule. If the peptide chains are spacedrelatively close on a surface, then the antibody can span two chains andthe observed effective binding constant may be greater than themonovalent value. Estimates of the surface density of the reactivepeptide chains on the surface suggest that this is likely to occur. Inaddition, the situation here is even more complex because a secondbivalent antibody was used to detect the initial binding of D32.39.

[0188] With the size and position of the epitope thus determined, thepresent invention was used to examine novel substitutions in the RQFKVVTpeptide sequence. The methods of the invention allowed systematicreplacement at each position in the lead sequence by other amino acids.A schematic of the substitutions is shown in Table 4, where X representsthe position undergoing substitution. When X is the 20 L-amino acids,the array comprised 140 peptides which were synthesized and screened forbinding. The masking technique illustrated in FIG. 1 with a translated,full-size mask, as shown in step 2 of FIG. 1A, was utilized. Preliminaryresults identified Q, K, V, and T as the residues most amenable tosubstitution. TABLE 4 Schematic of Illustrative Single Substitutionsinto the RQFKVVT Peptide Sequence XQFKVVT^(a) RXFKVVT RQXKVVT RQFXVVTRQFKXVT RQFKVXT RQFKVVX

[0189] These results demonstrate the application of a novel techniqueemploying both photolithography and solid phase peptide chemistry tocreate arrays of spatially-addressable chemical libraries. The abilityto screen simultaneously all the immobilized peptides for binding to abiological target allows one to generate powerful structure activityrelationship (SAR) databases. The use of novel building blocks as a toolto impart desirable physical properties into the arrays should aid inthe optimization of new lead compounds in the area of drug discovery.

[0190] B. Example

[0191] The example below illustrates aspects of one methodology for theformation of cyclic polymers. The method may be used to construct arraysof cyclic polymers according to the above methods.

[0192] Eight slides derivatized with NVOC-aminocaproic acid werephotodeprotected for ten minutes in 5 mM sulfuric acid/dioxane using 365nm light. After neutralization of the surface, six of the slides wereexposed to 0.1 M BOP activated NVOC-Glu(O-t-butyl)-OH, while theremaining two slides were exposed to 0.1 M BOP activated NVOC-Glu-OFm.The first six slides were divided into three groups and each group wasderivatized with either BOP activated Boc-Pro-Pro-Pro-Pro-OH,Boc-Ala-Ala-Ala-Ala-OH, or Boc-Ala-Gly-Gly-Gly-OH. The second two slidesfrom above were derivatized with BOP activated Boc-Val-Val-Val-Val-OH.This gave four pairs of slides, each with a pentapeptide on the surfacewith a side chain carboxyl (still protected) with which to cyclize. Eachslide was deprotected with TFA to remove the Boc and t-butyL groups(from both the amino terminus and the masked carboxyl group), and thenthe two slides with Fm as a protecting group were treated withpiperidine to unmask the carboxyl group.

[0193] A sixteen-well template was placed on each slide in order tophysically segregate different regions of the surface and one member ofeach pair was warmed (either to 41 or 44° C.) while the second memberwas kept at 20° C. during the following reactions. Each well of thetemplate was treated with either a 0.1 M solution of activator orsolvent for 4.5 hours. The activators were BOP, HBTU, anddiphenylphosphoryl azide (DPPA). After the specified time, the wellswere washed and the templates removed. The slides were stained with a 10mM solution of a 9:1 mixture of phenyl isothiocyanate (PITC):fluorescein isothiocyanate (FITC). The slides were washed and scannedfor fluorescence using a confocal microscope.

[0194] Cyclization of the peptides was expected to result in the loss ofreactivity of the terminal amine, and hence, the loss of fluorescenceintensity. Cyclization efficiency was measured as the decrease influorescence intensity for the peptides that had been treated with anactivator as compared to untreated peptides. Cyclization was found tooccur readily in all cases. The activators BOP and HBTU were found to bemore effective than DPPA. Temperature had little effect on thecyclization efficiency.

[0195] V. Conclusion

[0196] The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example, whilethe invention is illustrated primarily with regard to peptide,oligosaccharide and nucleotide synthesis, the invention is not solimited. By way of another example, while the detection apparatus hasbeen illustrated primarily herein with regard to the detection of markedreceptors, the invention will find application in other areas. Forexample, the detection apparatus disclosed herein could be used in thefields of catalysis, DNA or protein gel scanning, and the like. Thescope of the invention should, therefore, be determined not withreference to the above description, but instead should be determinedwith reference to the appended claims along with their full scope ofequivalents.

What is claimed is:
 1. A method of synthesizing an array of moleculescomprising the steps of: identifying a target molecule having a monomersequence complementary to a receptor of interest; and synthesizing anarray of molecules on a substrate by the steps of activating predefinedregions of said substrate and coupling selected monomers to saidsubstrate to form an array of molecules, said array of moleculescomprising at least first and second groups of molecules wherein: i)said first group has a first subsequence of monomers common to saidtarget molecule, and a first position in said target moleculesystematically substituted with members of a basis set of monomers, andii) said second group has a second subsequence of monomers common tosaid target molecule, and a second position in said target moleculesystematically substituted with said members of said basis set ofmonomers.
 2. The method as recited in claim 1 further comprising thestep of identifying which, if any, of said molecules binds to areceptor.
 3. The method as recited in claim 1 wherein the synthesizingstep comprises the steps of: performing a first series of activation andcoupling steps in which each of said first coupling steps activates andcouples monomers in said target molecule in a selected region of saidsubstrate, said selected regions each comprising a portion of a regionpreviously activated; and performing a second series of activation andcoupling steps in which each of said second coupling steps adds adifferent monomer from said basis set to a portion of the moleculesformed in each of said selected regions.
 4. The method as recited inclaim 1 wherein said activating steps comprise the step of irradiatingsaid substrate.
 5. The method as recited in claim 3 wherein saidactivation steps comprise the step of irradiating said substrate.
 6. Themethod as recited in claim 3 further comprising the steps of performinga third series of activation and coupling steps in which each of saidsteps couples monomers in said target molecule in a selected region ofsaid substrate.
 7. The method as recited in claim 1 wherein said arrayof molecules comprise: a23 b23 1a3 1b3 12a 12b wherein: 1, 2, and 3 aremonomers in said target molecule; and a and b are monomers in said basisset of monomers.
 8. The method as recited in claim 1 wherein said targetmolecule is selected from the group consisting of peptides,oligonucleotides, and oligosaccharides.
 9. The method as recited inclaim 1 wherein said target molecule is a peptide.
 10. The method asrecited in claim 1 wherein said target molecule is an oligonucleotide.11. The method as recited in claim 5 wherein: said first series ofirradiating steps uses a first mask having at least first and secondtransparent regions spanning a vertical portion of said substrate; andsaid second series of irradiating steps uses a mask having ahorizontally oriented transparent region spanning portions of saidsubstrate corresponding to said first and second transparent regions.12. The method as recited in claim 1 further comprising the steps of:contacting siad array with a receptor; determing which of the moleculesin the array are complementary to said receptor by identifying wheresaid receptor has bound to said array.
 13. An array of cyclic polymerson a substrate, said polymers having N monomer positions, said arraycomprising at least N different substrate sites, said substrate sitescomprising said cyclic polymers coupled thereto, said cyclic polymerscomprising common monomer sequences but coupled to said substrate via adifferent one of said monomer positions in each of said different sites.14. An array as recited in claim 13 wherein said polymers are coupled tosaid substrate via a common tether molecule.
 15. An array as recited inclaim 14 wherein said common tether molecule substitutes for a differentone of said monomers in said common monomer sequence within each of saiddifferent sites.
 16. An array as recited in claim 14 wherein said commontether molecule is added to said common monomer sequence at a differentposition is said common monomer sequence within each of said differentsites.
 17. An array as recited in claim 14 wherein said monomers areselected from the group consisting of amino acids and nucleic acids. 18.An array as recited in claim 14 wherein said array further comprisesmono-substituted polymers from said common monomer sequence, each ofsaid mono-substituted polymers coupled to said substrate in differentsites via a different monomer position in different sites in said array.19. Apparatus for screening cyclic polymers for biological activitycomprising an array as recited in claim 13, and further comprising:means for exposing said array to a receptor of interest; and means fordetecting positions on said array where said receptor binds to one ormore of said sites, whereby cyclic polymers complementary to saidreceptor may be identified.
 20. An array as recited in claim 16 wherein:said polymers are peptides; and said tether molecule adds 2 covalentlybonded atoms to a backbone chain of said polymers.
 21. An array asrecited in claim 20 wherein said tether molecule forms a disulfide bondin said cyclic polymers.
 22. An array as recited in claim 14 whereinsaid tether molecule is glutamic acid and said polymers are peptides.23. A method of forming an array of cyclic polymers on a substratecomprising: coupling a first monomer to a terminus of polymer moleculeson at least a first site on a substrate, without coupling said firstmonomer to polymer molecules in at least a second site on saidsubstrate; coupling a common monomer sequence to said polymer moleculesin said first and second sites; coupling a second monomer to polymermolecules in at least said second site to form first linear polymers insaid first sites and second linear polymer molecules in said secondsites; and forming cyclic polymers from said first and second linearpolymers, whereby cyclic polymers with different rotational orientationsare formed at said first and second sites.
 24. The method as recited inclaim 23 wherein said step of coupling a second monomer comprises thesteps of: irradiating said second site with light to remove a protectinggroup from polymer molecules in said second site; and exposing saidsubstrate to said second monomer to said substrate whereby said secondmonomer is coupled to to polymer molecules in said second site.
 25. Themethod as recited in claim 24 further comprising the steps of: exposingsaid polymers to a receptor; determining which of said polymers iscomplementary to said receptor by determining where said receptor hasbound to said substrate.
 26. The method as recited in claim 23 whereinsaid polymers are peptides.
 27. The method as recited in claim 24wherein said polymers are oligonucleotides.
 28. A method of forming anarray of molecules comprising the steps of: identifying a targetmolecule having N monomer positions; and forming an array of moleculeson a substrate in which said monomer positions are systematicallysubstituted with a basis set of monomers by the steps of: i) formingtruncated target molecules having lengths from 1 to N−1 at predefinedregions of said substrate; ii) coupling each of said members of saidbasis set to each of said truncated target molecules in predefinedregions of said substrate; and iii) coupling remaining monomers in saidtarget molecule to said truncated target molecules, thereby forming anarray of molecules in which each monomer position in said targetmolecule is systematically substituted with each member of said basisset of monomers.
 29. The method as recited in claim 28 wherein saidmonomers are amino acids.
 30. The method as recited in claim 28 whereinsaid monomers are nucleotides.
 31. The method as recited in claim 28wherein said step of forming truncated target molecules comprises thesteps of: removing groups from at least two predefined regions of saidsubstrate; coupling a first monomer in said target molecule to said atleast two predefined regions, said first monomer comprising a protectinggroup; removing protecting groups in one of said two predefined regionsof said substrate; and coupling a second monomer in said target moleculein said one predefined region.
 32. The method as recited in claim 31wherein the step of coupling each of said members of said basis setcomprises the steps of: removing protecting groups in first portions ofboth of said two predefined regions; coupling a first monomer from saidbasis set in said first portions of said two predefined regions;removing protecting groups in second portions of both of said twopredefined regions; and coupling a second monomer from said basis set insaid second portions of said two predefined regions.